The present invention concerns fluorescent substrates and fluorophores produced therefrom useful in fluorescent detection methods. Measurement of the fluorescence produced by these fluorophores is indicative of the presence and the amount of fluorophore in a sample.
Immunoassays employing the unique specificity of antibodies for their counterpart antigens at the molecular binding level have been demonstrated to be extremely valuable methods of analysis to determine the presence or absence of one or more components. The specificity of an antibody for its coupling partner, i.e., counterpart antigen, permits the detection of very small concentrations of specific antigens or antibodies in the presence of considerable other biomaterials (proteins, lipids, sugars, etc.) as usually found in body fluids or other biological samples.
In the past several decades, one of the more useful approaches employed to take advantage of this specificity utilizes the labeling or tagging of either the antibody or the antigen, depending upon the analyte sought in the sample. It is the label or tag which is detected in making a determination with respect to the analyte sought. Exemplary of labels that may be employed are radioactive labels which can be detected in scintillating or gamma counters, fluorochromes which can be detected in fluorometers, luminescent labels which spontaneously emit light and can be detected by light detectors, and enzymes labels which in themselves are not detectable but which, when allowed to interreact with other "substrate" molecules, produce changes in the substrate which can be detected. The advantage of the enzyme label is the fact that a single enzyme molecule can alter many tens of thousands of substrate molecules per minute, and this translates into enhanced sensitivity.
The use of enzyme labels in an assay system has been referred to as an enzyme-labelled immuno-sorbant assay, which for brevity is sometimes referred to as an ELISA technique. Such assays are the subject of a number of U.S. patents, including the following: U.S. Pat Nos. 3,654,090; 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,016,043; RE 29,169; U.S. Pat. Nos. 4,152,411; 4,169,012; 4,228,240; 4,292,403; 4,331,761; 4,343,896; RE 31,006.
In a typical ELISA technique, a binding partner for the substance to be determined is reduced to an insolubilized form, as by adsorption of the binding partner on the wall of a plastic tube or other adsorbant, i.e., reactive surface. The substance to be determined is reacted with the insolubilized binding partner and the liquid phase of the reaction is subsequently separated. The solid phase is thereafter reacted with a determined amount of coupling partner of the substance to be determined, which coupling partner has been tagged with an enzyme. A coupled product results, and to this product there is added a liquid substrate reactable with the enzyme labels or tags of the coupling partner. The presence of resulting products are usually determined by viewing the color of the liquid phase in the final reaction mixture. Additionally, a chemical stopper is often added to the reaction mixture to inhibit further enzymatic reaction. In a chromogenic detection system, this chemical stopper often produces a change in the resulting product which causes a resultant change in color for easier visual read out. Many regard the chemical stopper, often acid or alkali, not so much as a stopper but rather as a color developer or enhancer.
To obtain greater sensitivity in an enzyme immunoassay, an end product measurable by fluorescent techniques would be preferred (see K. H. Milby in "Enzyme-Mediated Immunoassay", T. T. Ngo and H. M. Lenhoff, eds. Plenum Press, New York, pp. 325-341, 1985).
Fluorescence is a process by which a molecule that is excited by light of a given wavelength emits light at a longer wavelength. The intensity of light emission from a collection of fluorescent molecules depends on:
(1) the intensity of the excitation light source; PA0 (2) the amount of light absorbed, which according to Beer's Law, depends directly on the concentration of the fluorescent molecules present; and PA0 (3) the efficiency with which the fluorescent molecules convert absorbed light into emitted light (the fluorescence quantum yield of the molecule).
If a constant level of excitation light intensity is maintained, emitted light intensity is directly proportional to the number of fluorescent molecules present.
Fluorescence in general is considered to be up to 100 times more sensitive than spectrophotometric techniques or other color-change detection techniques. The use of .beta.-D-galactosidase enzyme and its fluorogenic substrate 4-methylumbelliferyl .beta.-D-galactoside has been cited in the literature as an example of this type of assay (Ishikawa E., Imagawa M., & Hashids S., "Ultrasensitive Enzyme Immunoassay Using Fluorogenic, Lumenogenic, Radioactive and Related Substrates and Factors to Limit Sensitivity", J. Biochem 73, 1319-1321, 1973).
By using fluorescent molecules as labels in immunoassays, either directly attached to an antibody or antigen (called fluorescence immunoassay, or FIA) or as a fluorogenic substrate to detect an enzyme attached to antibody or antigen (called enzyme-linked fluorescence immunoassay, EFIA, or F-ELISA, fluorescence enzyme-linked immunosorbent assay), reagent systems and instruments have been developed which allow quantitative detection of analyte levels with high efficiency and sensitivity.
FIA has the advantages of simplicity of procedure, immediate end point measurement, and excellent reproducibility (precision) of results. EFIA offers greater sensitivity than FIA, since the catalytic activity of each enzyme label can produce up to 10,000 or more fluorophores by proper choice of fluorophore precursor molecule, i.e., fluorogenic substrate. Among the fluorescent molecules most frequently used in immunochemistry are fluorescein, the rhodamines, certain coumarin (umbelliferone) derivatives, and most recently, the phycobiliproteins.
Fluorescein is currently the label of choice in FIA. Its physical properties illustrate the factors important in a fluorophore label. Fluorescein is a very efficient emitter of light, with a fluorescence quantum yield of 0.3 to 0.95 (vs. the maximum possible of 1.0) when conjugated (attached) to an antibody or antigen. It is also an efficient absorber of light, with a molar extinction coefficient [related to the probability of absorbing a photon that strikes the fluorescein] of 70,000.
Fluorescein is stable and undergoes little degradation (photobleacing or photolability) when exposed to light. Techniques for labelling antibodies or protein antigens with fluorescein are relatively simple, but are not easy to control. Fluorescein's fluorescence efficiency is affected very little by temperature, but only the dianion form of the molecule, which exists above pH 8, emits strongly, i.e., fluorescence efficiency varies considerably with pH except at pH 8 or above.
On the negative side, fluorescein emits at 525 nm, with most efficient excitation at 490 nm. Its fluorescence signal is thus subject to the following types of interference which limit its effective sensitivity: (1) overlap by endogeneous fluorescence, i.e. light emitted by sustenances such as bilirubin and hemoglobin, which are normally found in serum samples; (2) quenching, e.g., by bilirubin; and (3) inner filter effects, i.e. preferential absorption of incident light by other molecules such as hemoglobin. Its small Stoke's shift (30 nm) also leads to significant background scattering, which must be eliminated by using complex, dedicated (and thus expensive) filter systems and/or by sacrificing certain efficiencies in instrument design, e.g., off-peak excitation or emission detection. Fluorescein also self-quenches (inhibits its own fluorescence) if molecules are brought into close proximity, so multiple labeling even of large molecules like antibodies is of limited use, even though fluorescein is a small molecule.
Other fluorophores also have certain advantages and drawbacks. The rhodamines fluoresce at long wavelengths, but have poor Stoke's shifts, relatively low fluorescence quantum yields, and are extremely sensitive to pH and other environmental effects. The coumarins have relatively large Stoke's shifts and high extinction coefficients but low quantum yields, are pH sensitive, and their emission is above 500 nm, i.e., subject to endogenous sample interference.
The biliproteins have very high extinction coefficients, long wavelength emission, and in the case of R-phycoerythrin, good Stoke's shifts. They also display high quantum yields, and their emission is relatively insensitive to environment within the pH range of 5.5 to 9. These molecules are large proteins which must be isolated, e.g., from red algae. One such biliprotein, R-phycoerythrin, can provide sensitivity 10-20 times greater than that afforded by fluorescein in FIA systems. Nevertheless the large size and high molecular weight of these molecules, particularly of R-phycoerythrin, place significant limitations on applications of these molecules in immunoassays because of effects on reaction kinetics; e.g., they can't be used to replace fluorescein in fluorescence polarization or in hapten assays with competitive binding formats.